Biological Sciences Research Highlights

Faster, more sensitive, more effective alternative for probing reversible modifications

Chemistry principles for selective conversion of redox modifications and the analytical workflow of redox proteomics, as developed by PNNL scientists. Redox proteomics is a powerful tool to study the mechanism of cellular regulation in various organisms such as bacteria, plants, and mammalian cells or tissues, and to identify novel functional regulators. Enlarge Image.

Results:
In all
organisms, reduction-oxidation—or "redox"—regulation is essential for many
biological processes, including metabolism, gene expression, and environmental
and stress responses. Key to understanding these processes is being able to determine how the post-translational modifications (PTMs) of proteins, which occur in this case on their cysteine residues, contribute to creating "redox switches" capable of regulating protein functions and protein-protein interactions. Researchers at Pacific Northwest National Laboratory (PNNL) recently developed an efficient method for enriching and quantitatively analyzing PTMs of cysteine residues of proteins using a commercially available resin.

The procedure was effective and
efficient in enriching—or delivering higher quantities—of pertinent cysteine
thiols for analysis. It's anticipated the team's advance will offer broad
applications in biological studies by serving as a general enrichment strategy
for multiple types of reversible cysteine modifications.

Why
It Matters: Research has shown cysteine thiols in
proteins frequently participate in enzymatic reactions and are subjected to a
variety of covalent PTMs, or changes that can significantly impact protein behavior
and functioning.

"The biological significance of several
types of reversible cysteine PTMs has increasingly been recognized, as they
have been found to modulate protein function in a variety of biological
pathways," said Dr. Jia Guo, a researcher from PNNL's Integrative Omics group.

Enrichment of cysteine thiols
traditionally has been achieved using the biotin-switch technique (BST), a
method that has greatly advanced knowledge in the field. However, BST is labor intensive.
Also, there may be instances where the technique captures the wrong target
subjects during the enrichment process.

"Our resin-assisted approach is simpler
because it facilitates the direct covalent capture of thiol-containing proteins,"
Guo explained. "Subsequent steps in the process negate the need for sample
cleanup prior to mass spectrometer analysis, saving a significant amount of time."

Considering that much remains to be
learned about cysteine-based reversible modifications, the PNNL-developed innovation
is a key step toward more rapidly expanding knowledge in this field.

Methods:
The team tackled
this challenge by integrating chemical derivatization, specific resin-based
enrichment, and advanced mass spectrometry to achieve quantitative measurements
of specific types of modifications. In its inquiry, the researchers employed S-nitrosylation-modified
peptides enriched from mouse muscle.

The technique's
workflow starts with direct capture of derived free thiol-containing proteins
onto the thiopropyl Sepharose 6B resin, followed
by the breaking of remaining protein chains into smaller chains on the resin, then
multiplex isobaric labeling—also on the resin—and, finally, extraction of the
captured peptides. The labeled peptides are subjected to liquid-chromatography-mass
spectrometry to identify specific modified cysteine residues and quantify
reversible modifications.

Although
there are potential limitations in the method, the research team believes the
resin-assisted approach represents clear progress in identifying novel cysteine
sites sensitive to redox or other reversible modifications.

"Our
approach can help build knowledge of cysteine-based reversible modifications by
helping to overcome challenges associated with isolating, identifying and
quantifying the modifications," Guo said.

What's
Next: Researchers are applying the
methodologies to studies of redox regulation and signaling in cyanobacteria
relevant to biofuel research. The approaches also are being used in a mammalian
tissues inquiry examining the role of redox regulation in oxidative
stress-related diseases.

Acknowledgments:

Sponsors:
U.S. Department
of Energy (DOE) Early Career Research Award, DOE Office of Biological and
Environmental Research's Genomic Science Program through the PNNL Pan-omics
Scientific Focus Area, and the National Institutes of Health Director's New
Innovator Award Program.